Antibody microarray analysis of cell surface antigens on CD4+ and CD8+ T cells from HIV+ individuals...

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Open AcceResearchAntibody microarray analysis of cell surface antigens on CD4+ and CD8+ T cells from HIV+ individuals correlates with disease stagesJing Qin Wu1, Bin Wang1, Larissa Belov2, Jeremy Chrisp2, Jenny Learmont3, Wayne B Dyer3, John Zaunders4, Anthony L Cunningham1, Dominic E Dwyer5 and Nitin K Saksena*1
Address: 1Retroviral Genetics Division, Center for Virus Research, Westmead Millennium Institute, Darcy Road, Westmead, NSW 2145, Sydney, Australia, 2Medsaic Pty Ltd, Suite 145, National Innovation Centre; Australian Technology Park, Garden Street, Eveleigh, NSW 1430, Sydney, Australia, 3Viral Immunology Laboratory, Australian Red Cross Blood Service, Clarence Street, NSW 2000, Sydney, Australia, 4Center for Immunology, Darlinghurst, NSW, Sydney, Australia and 5Department of Virology, ICPMR, CIDM Labs, Westmead Hospital, Westmead, NSW 2145, Sydney, Australia

Email: Jing Qin Wu - [email protected]; Bin Wang - [email protected]; Larissa Belov - [email protected]; Jeremy Chrisp - [email protected]; Jenny Learmont - [email protected]; Wayne B Dyer - [email protected]; John Zaunders - [email protected]; Anthony L Cunningham - [email protected]; Dominic E Dwyer - [email protected]; Nitin K Saksena* - [email protected]

* Corresponding author

AbstractBackground: Expression levels of cell surface antigens such as CD38 and HLA-DR are related to HIVdisease stages. To date, the immunophenotyping of cell surface antigens relies on flow cytometry, allowingestimation of 3–6 markers at a time. The recently described DotScan antibody microarray technologyenables the simultaneous analysis of a large number of cell surface antigens. This new technology providesnew opportunities to identify novel differential markers expressed or co-expressed on CD4+ and CD8+T cells, which could aid in defining the stage of evolution of HIV infection and the immune status of thepatient.

Results: Using this new technology, we compared cell surface antigen expression on purified CD4+ andCD8+ T cells between 3 HIV disease groups (long-term non-progressors controlling viremia naturally;HIV+ patients on highly active antiretroviral therapy (HAART) with HIV plasma viral loads <50 copies/ml;and HIV+ patients with viremia during HAART) and uninfected controls. Pairwise comparisons identified17 statistically differential cell surface antigens including 5 novel ones (CD212b1, CD218a, CD183, CD3epsilon and CD9), not previously reported. Notably, changes in activation marker expression were morepronounced in CD8+ T cells, whereas changes in the expression of cell membrane receptors for cytokinesand chemokines were more pronounced in CD4+ T cells.

Conclusion: Our study not only confirmed cell surface antigens previously reported to be related to HIVdisease stages, but also identified 5 novel ones. Of these five, three markers point to major changes inresponsiveness to certain cytokines, which are involved in Th1 responses. For the first time our studyshows how density of cell surface antigens could be efficiently exploited in an array manner in relation toHIV disease stages. This new platform of identifying disease markers can be further extended to studyother diseases.

Published: 26 November 2007

Retrovirology 2007, 4:83 doi:10.1186/1742-4690-4-83

Received: 24 August 2007Accepted: 26 November 2007

This article is available from: http://www.retrovirology.com/content/4/1/83

© 2007 Wu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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BackgroundHIV infection leads to characteristic alterations in the sub-set composition of circulating CD4+ and CD8+ T lym-phocytes. The activation marker CD38, in particular, andits level of expression on CD8+ T cells is a marker that isstrongly associated with immune activation, particularlyduring primary HIV-1 infection and progression to AIDS,respectively [1-3]. Furthermore, decreased expression ofCD38 on CD8+ T cells is highly correlated with the effec-tiveness of antiretroviral therapy [4-8] and lack of activa-tion and expression of CD38 and HLA-DR on CD4+ Tcells correlates with long-term non-progression [9]. Theenumeration of CD4+ T-lymphocytes by flow cytometry isused routinely in the clinical management of HIV-infectedindividuals to monitor the severity of immunodeficiencycaused by HIV, and this acts as a basis for commencingHAART and prophylaxis for Pneumocystis carinii pneumo-nia [10]. However, more information on the progressionto immunodeficiency may be found in the detailed subsetcomposition of CD4+ and CD8+ T cells, but this is cur-rently restricted to research studies.

To date, the immunophenotyping of CD antigens relieson flow cytometry. Although very reliable, the flow

cytometry only allows estimation of 3–6 markers in agiven assay. The recently developed antibody microarraytechnology enables the simultaneous analysis of a largenumber of cell surface antigens on a single chip. This newtechnology may permit the identification of novel differ-ential markers expressed or co-expressed on CD4+ andCD8+ T cells, which could aid in defining the stage of evo-lution of HIV infection and the immune status of thepatient [11]. This antibody microarray also has significantadvantages over gene expression microarray because itprofiles cells at the level of protein expression, rather thanrelying on quantifying mRNA expression levels. Thepower of this technology as an adjunct to flow cytometrywas recently highlighted by Woolfson et al. [12], who useda similar antibody microarray to demonstrate the conser-vation of unique cell surface antigen mosaics in cryopre-served PBMCs from HIV+ individuals.

Here, we have used an antibody microarray constructedon the surface of a nitrocellulose coated slide to simulta-neously analyze 135 different cell surface antigens (128cluster of differentiation antigens plus 7 other surfaceantigens) on peripheral blood CD4+ and CD8+ T cell sub-sets from HIV+ and HIV- individuals. A comparison of

Table 1: Patient clinical details of viral load, CD4+ and CD8+ T cell counts at the time of sample collectiona

Patient Date Age CD4 counts (cells/µl) CD8 counts (cells/µl) Viral Load (copies/ml) Disease Group

1 20/06/06 55 380 790 <50 BDL2 21/06/06 23 355 492 <503 27/06/06 61 691 1538 <504 27/06/06 42 425 721 <505 02/08/06 62 1037 607 <506 02/08/06 52 750 368 <507 23/08/06 52 799 449 <508 03/09/06 49 371 1818 <509 12/09/06 62 458 206 <5010 13/09/06 39 901 870 <5011 20/09/06 61 782 1936 <5012 02/08/06 54 292 2989 352 VIR13 21/06/06 38 560 980 4950014 16/08/06 39 500 2064 3260015 15/08/06 57 461 2306 4690016 29/08/06 41 390 463 56717 07/09/06 61 170 529 34518 12/09/06 43 251 642 170019 15/09/06 50 265 170 10520 20/09/06 38 231 1289 4690021 03/10/06 45 286 1144 38822 28/06/06 58 690 650 <50 LTNP23 28/06/06 50 817 513 <5024 18/07/06 78 880 860 <5025 21/07/06 58 760 1800 <5026 03/10/06 32 1121 790 128

a Plasma viral load was measured using the Quantiplex HIV RNA3.0 (Chiron bDNA) assay with a lower limit of detection of 50 HIV-1 copies/ml (Chiron Diagnostics, Halstead, United Kingdom).



CD4+ and CD8+ T cells purified from peripheral blood ofthree different HIV-infected patient groups (Table 1, HIV+therapy naïve long-term non-progressors with high CD4+and CD8+ T cell counts; HIV+ patients on HAART withplasma viremia below detectable levels; and viremicpatients on HAART) and HIV seronegative individualsshowed 17 statistically differential cell surface antigens, 5of which were novel. Furthermore, we demonstrate thatchanges in the expression of activation markers were morepronounced in CD8+ T cells, whereas changes in theexpression of cell membrane receptors for cytokines andchemokines were more pronounced in CD4+ T cells.

ResultsThe pairwise comparisons of groups identified 17 statisti-cally differential cell surface antigens, of which,CD212b1, CD218a, CD183, CD3epsilon and CD9 havenot been previously reported in the context of HIV dis-ease.

Discriminatory antibodies for CD4+ T cellsThe signature pattern for each group and dot pattern fromwhich the raw data were derived is shown in Figure 1 and2, respectively. In pairwise comparisons, CD4+ cells fromthe HIV+ individuals differed from those of the NEGgroup, as shown by the significant upregulation of CD71,CD212b1, HLA-DR, CD95, CD57 and CD11b on theCD4+ cells of one or more of the HIV+ groups, as shownin Table 2. Although several other antigens showedincreased (CD218a and CD86 in VIR) or decreased(CD27 in LTNP, CD45RA in BDL and CD28 in VIR)expression compared to the NEG group, these did notalways reach statistical significance, with p values rangingfrom 0.0540 to 0.0744.

Average CD11b and CD95 expression also increased in all3 HIV+ groups compared to NEG individuals, but thisupregulation reached significance only in VIR, the p valuein BDL and LTNP ranging from 0.0589 to 0.0752. Varia-bility in antigen expression within the latter groups maycontribute to this lack of significance, though the differ-ences may reach significance with increased group sizes.

CD71, CD218a and CD54 were upregulated in both theLTNP and the VIR groups compared with BDL, with p val-ues of < 0.05 for all except CD54 in the LTNP-BDL com-parison (p = 0.0695). LTNP-CD4 differed from BDL and/or VIR groups in that CD71, HLA-DR, CD38, CD3epsilonand CD183 were significantly upregulated.

Discriminatory antibodies for CD8+ T cellsThe signature pattern for each group and representativedot pattern from which the raw data were derived areshown in Figure 3 and 4, respectively. In pairwise compar-isons, CD8+ cells from HIV+ individuals differed from the

NEG group, as shown by the significant upregulation ofHLA-DR, CD57, CD11c, CD45RO and CD95 in one ormore of the HIV+ groups, with all 3 HIV+ groups showingan increase in average HLA-DR, CD57, CD45RO andCD95 expression (Table 3). There was significant down-regulation of CD9 (in VIR) and CD27 (in LTNP), whileincreases in CD212b1 (in LTNP and VIR) did not reachsignificance (p = 0.0745–0.0776). CD38 was significantlyupregulated in the VIR group compared with BDL andNEG groups. LTNP differed from BDL and VIR groups inthat CD8+ cells showed higher expression of CD11c,CD16 and CD56, all differences being statistically signifi-cant except CD56 in the LTNP-BDL comparison (p =0.0871).

DiscussionTo date the analysis of cell surface antigens on peripheralblood lymphocytes of HIV infected individuals has beenlargely carried out with whole PBMC using flow cytome-try. In this study we used DotScan antibody microarraytechnology (Medsaic Pty. Ltd, Sydney, Australia) to simul-taneously analyze CD4+ and CD8+ T cell subsetsobtained from HIV+ individuals at different stages of HIVdisease for the expression of 135 different cell surface anti-gens. For the first time, our study shows how the immu-nophenotype of diverse blood cell types (in this caseCD4+ and CD8+ T cells) can be exploited to study variousHIV disease stages using antibody microarray technology.Our analyses identified 17 statistically significant (p <0.05) differentiating CD antigens distributed betweenCD4+ and CD8+ T cells. Of these, 11 (CD71, CD212b1,HLA-DR, CD57, CD95, CD11b, CD38, CD3epsilon,CD218a, CD54 and CD183) were differential for CD4+ Tcells, while 10 (CD9, CD11c, CD16, CD38, CD27,CD45RO, CD56, CD57, CD95 and HLA-DR) for CD8+ Tcells. Among these, CD212b1, CD218a, CD183,CD3epsilon and CD9 have not been previously describedin relation to HIV disease.

For the activation markers, the results are in accordancewith previous studies using flow cytometry, confirmingthe utility of this new technology. As previously reported[13,14], HLA-DR and CD38 were significantly upregu-lated on both CD4+ and CD8+ T cells during HIV infec-tion. On CD4+ T cells, we observed significantupregulation of HLA-DR in LTNP comparison to BDL andNEG groups, suggesting that the level of activation ofCD4+ T cells from the BDL group was reduced by HAARTto a level similar to the NEG group. For CD38, a signifi-cant increase was detected in the LTNP compared to theBDL group. This may be due to the intermediate expres-sion of CD38 on naïve CD4+ T cells, but this was not con-firmed as this study did not differentiate between naïveand memory CD4+ T cells. On CD8+ T cells, significantupregulation of HLA-DR was observed in all HIV+ groups,




Antibody response charts of CD4+ T cellsFigure 1Antibody response charts of CD4+ T cells. The bar charts represent the average immunophenotypes, or signatures, for each of the disease categories. Asterisks show the antigens which were significantly up-or down-regulated in paired comparisons of the disease groups. Labeling on the x-axis refers to monoclonal antibodies and their specificities against the corresponding anti-gens and the y-axis the binding densities.


while CD38 upregulation was seen in two HIV+ groups(LTNP and VIR compared to the NEG group) and the pair-wise comparison of VIR versus BDL group, which is in fullaccordance with previous studies showing CD38 expres-sion on CD8+ T cells is a marker associated with HIV dis-ease progression [1,2]. The increased expression ofCD45RO on CD8+ T cells during HIV infection has alsobeen well documented [15], supporting our observationthat increases in CD45RO expression were significant, orclose to significant, on CD8+ T cells in all 3 HIV+ groups.However, CD45RO modulation was not observed onCD4+ T cells, confirming flow cytometry data [13], whichalso showed that the proportion of CD4+ T cells express-ing CD45RO remained relatively unchanged. Takentogether, it appears that for the activation markers men-tioned above, significant changes in expression were morepronounced on CD8+ than CD4+ T cells.

In addition, CD27 was significantly downregulated onCD8+ T cells in the LTNP group compared to the NEGgroup. It has been suggested in one study that HIV-specificCD8+ T cells that have differentiated to the CD27- stageare related to the delayed disease progression [16], whileanother study has also observed a similar trend [17]. TheCD8+ T cells used in our study were not selected for HIV-specificity, and hence the LTNP status appears to berelated to a general downregulation of CD27.

We also observed significantly increased CD95 expressionon both CD4+ and CD8+ T cells in the VIR group.Although partially elevated CD95 levels were alsoobserved in BDL on HAART and LTNP groups, theincreases were not significant, which is consistent withreduced immune activation in patients with reduced viralreplication. Increased Fas-receptor (CD95) expression onCD4+ and CD8+ lymphocytes has previously been dem-onstrated in a large group of HIV-1-infected patients when

Composite dot scan patterns of antibody binding for CD4+ T cellsFigure 2Composite dot scan patterns of antibody binding for CD4+ T cells. Half of a duplicate array was shown with the alignment dots "A" at left, top and bottom. Alignment dots are a mixture of CD44 and CD29 antibodies. (A) The key for CD antigens on the DotScan array, (B) NEG, (C) BDL, (D) VIR and (E) LTNP. Binding patterns shown here are representatives from each group. They may not fully reflect all the significant antigens from statistical analysis because of individual variability in antigen expres-sion.



compared against normal controls [18], and evidence alsosuggests that poor responders to antiretroviral therapymay have significantly higher CD95 expression [19].These findings, together with our results, suggest that sig-nificantly increased CD95 expression may relate toantiretroviral therapy failure.

The most notable feature for the activation markers wassignificant downregulation of CD9 expression on CD8+ Tcells in the VIR group compared to the NEG group. CD9belongs to a transmembrane protein family known as the

tetraspanin family. It has been proposed that these pro-teins act as scaffolding proteins by laterally organizing cel-lular membranes via specific associations with each otherand distinct integrins. A recent study has shown that thetetraspanin-enriched microdomains on the cell mem-brane can function as gateways for HIV egress [20]. Theoverall functional relevance of this antigen in the contextof HIV disease requires further investigation.

It has been suggested that CD57 is a marker for replicativesenescence [21,22]. We found a significant increase ofCD57 expression on CD8+ T cells in 2 of the 3 HIVinfected groups (BDL and VIR) compared with NEGgroup, while on CD4+ T cells, only the VIR group showed

Table 3: Discriminatory antibodies for CD8+ T cellsa

Discriminatory Antibody Up(+)/Down(-) P Value

BDL vs. NEGCD57 + 0.0272CD45RO + 0.0307HLA-DR + 0.0315CD95 + 0.0538LTNP vs. NEGHLA-DR + 0.0039CD11c + 0.0072CD38 + 0.0219CD27 PH - 0.0310CD95 + 0.0545CD57 + 0.0591CD16 + 0.0631CD212b1 + 0.0745CD45RO + 0.0953VIR vs. NEGCD57 + 0.0006CD45RO + 0.0067CD38 + 0.0091CD95 + 0.0091HLA-DR + 0.0190CD9 - 0.0268CD212b1 + 0.0776BDL vs. LTNPCD11c - 0.0049CD16 - 0.0093CD56 - 0.0871VIR vs. LTNPCD11c - 0.0017CD56 - 0.0121CD16 - 0.0308CD27PH + 0.0585CD9 - 0.0862VIR vs. BDLCD38 + 0.0124

aSix paired comparisons of CD markers on CD8+ cells providing significant discrimination between patient groups, with relative changes in CD antigen binding in the former vs. the latter denoted by "+" and "-" to indicate increase and decrease, respectively. Antigens that did not achieve statistical significance (p > 0.05) but have been previously reported in HIV disease context or have been found to be significant in other pair comparisons are also shown in italic.

Table 2: Discriminatory antibodies for CD4+ T cellsa

Discriminatory Antibody Up(+)/Down(-) P Value

BDL vs. NEGCD11b + 0.0599CD95 + 0.0619CD45RA - 0.0679LTNP vs. NEGCD71 + 0.0151CD 212 b1 + 0.0307HLA-DR + 0.0312CD27 PH - 0.0560CD11b + 0.0589CD95 + 0.0752VIR vs. NEGCD71 + 0.0032CD11b + 0.0227CD57 + 0.0307CD95 + 0.0349CD28 - 0.0540CD218a + 0.0732CD86 + 0.0744BDL vs. LTNPCD183 - 0.0105HLA-DR - 0.0270CD38 - 0.0316CD71 - 0.0385CD218a - 0.0470CD212 b1 - 0.0687CD54 - 0.0695VIR vs. LTNPCD183 - 0.0451CD3epsilon - 0.0489CD27 PH + 0.0829VIR vs. BDLCD71 + 0.0195CD218a + 0.0355CD54 + 0.0421

aSix paired comparisons of CD markers on CD4+ cells providing significant discrimination between patient groups, with relative changes in CD antigen binding in the former vs. the latter denoted by "+" and "-" to indicate increase and decrease, respectively. Antigens that did not achieve statistical significance (p > 0.05) but have been previously reported in HIV disease context or have been found to be significant in other pair comparisons are also shown in italic. The "PH" in CD27 PH distinguishes this Pharmingen antibody (clone M-T271) from the Immunotech antibody 27 IM (clone 1A4-CD27).




Antibody response charts of CD8+ T cellsFigure 3Antibody response charts of CD8+ T cells. The bar charts represent the average immunophenotypes, or signatures, for each of the disease categories. Asterisks show the antigens which were significantly up-or down-regulated in paired comparisons of the disease groups. Labeling on the x-axis refers to monoclonal antibodies and their specificities against the corresponding anti-gens and the y-axis the binding densities.


significantly increased expression. For HIV-specific CD8+cells, diminished proliferative capacity results in a mem-ory T cell population with reduced capacity to controlinfections [23]. Although we did not study pure HIV-spe-cific T cells, the significantly increased expression of CD57seems to be directly related to disease progression. In con-trast to CD57 which is linked to replicative senescence,the transferrin receptor CD71 has been reported as amarker for T cell proliferation [24]. DotScan analysisshowed that CD4+ T cells of LTNP and VIR groupsexpressed significantly higher levels of expression ofCD71 than that of the BDL group. Consistent with studiesshowing a significant increase in T cell turnover in HIVinfection [25-27], the upregulation of CD71 expression inthe LTNP group may indicate that the cells have recentlycycled. On the other hand, the upregulation of both CD57and CD71 in the VIR group may indicate the activationrather than the cell proliferation index [28]. Since CD71constitutively cycles from endosomes to the cell surface

and back again [29], the microarray was significantly bet-ter at detecting CD71 expression than flow cytometry.

Three cytokine receptors (CD183, CD218a, andCD212b1) were found to be significantly upregulated onCD4+ T cells in LTNP group, indicating a polarized Th1cell immune response in LTNP group in HIV infection.The CD4+ T cells of the LTNP group expressed higher lev-els of CD183 (CXCR3) than those of the BDL and VIRgroups. Since CD183 is reported to be preferentiallyexpressed on Th1 versus Th2 cells in peripheral blood[30,31] and to be found on a high percentage of CD4+ Tcells in type 1-dominated inflammatory processes [32],the upregulation of this protein in the LTNP group mayimply a polarization towards a Th1 immune response inLTNP group. CD218a (α chain of IL18R) was significantlyupregulated on the CD4+ T cells of LTNP and VIR groupscompared with the BDL group. CD212b1 (beta1 chain ofIL12R) was also significantly upregulated on the CD4+ Tcells of the LTNP group compared to the NEG group. A

Composite dot scan patterns of antibody binding for CD8+ T cellsFigure 4Composite dot scan patterns of antibody binding for CD8+ T cells. Half of a duplicate array was shown with the alignment dots "A" at left, top and bottom. Alignment dots are a mixture of CD44 and CD29 antibodies. (A) The key for CD antigens on the DotScan array, (B) NEG, (C) BDL, (D) VIR and (E) LTNP. Binding patterns shown here are representatives from each group. They may not fully reflect all the significant antigens from statistical analysis because of individual variability in antigen expres-sion.



previous study on the expression of cytokine receptors onlymphocytes from patients suffering from a disorder asso-ciated with raised Th1 cytokine production showed thatthe percentage of CD218a+ and CD212b1+ cells withinthe CD4+CD45RA+ subset is significantly higher in thesepatients than in healthy subjects [33]. By analogy, in HIVdisease, the upregulation of CD218a and CD212b1 inHIV disease may also indicate a chronically polarizedimmune response towards Th1 in LTNP group. Withregard to cytokine receptors, although previous studieshave shown that the level of CD127 (IL-7R) expressionrepresents a major difference between HIV+ subjects andcontrols [34-36], we did not observe any differencebetween groups. This is due to the lack of cell binding toCD127 antibody on the microarray, which may attributeto either low cell numbers of CD127+ or the low affinityof this antibody used.

With regard to cell signaling, CD3epsilon expression onCD4+ T cells was found to be significantly lower in theVIR group than in LTNP, which indicates that the expres-sion level of CD3epsilon can be used to differentiate VIRand LTNP groups. A previous study on HIV+ patients hasshown that the expression of CD3 complex (gamma,delta, epsilon) is downregulated on T cells compared tohealthy control [37], while our study is the first to relateone of the components of CD3 complex, CD3epsilon, toHIV disease status, yet the biological significance ofCD3epsilon expression in HIV disease requires furtherelucidation. Interestingly, the significant changes inexpression of cell membrane receptors mentioned above(CD183, CD218a, CD212b1 and CD3epsilon) were allobserved on CD4+ T cells, but not on CD8+ T cells.

Significant group-specific differences were also found for3 cell adhesion molecules (CD11b, CD11c and CD54),which may have significant implications for the patho-genesis of HIV disease, since adhesion molecules canaffect cell distribution, migration and immune response.CD11b was significantly upregulated on CD4+ T cells inthe VIR group compared with the NEG group. Thisincrease was also seen in the BDL and LTNP groups, butwas not statistically significant. Although there is evidencefor increases in CD8+/CD11b+ T cells during progressionof HIV infection in asymptomatic patients [38], we are notaware of any previous reports of modulations in CD11bexpression on CD4+ T cells of HIV+ patients. CD11c,which has mainly been studied in relation to dendriticcells in HIV disease, was found to be expressed at signifi-cantly higher levels on CD8+ T cells in the LTNP groupthan in the other 2 HIV+ groups. Although the upregula-tion of CD11c after HIV-1 infection has been reported atmRNA levels [39], it has not previously been documentedat protein levels. CD54, also known as intercellular adhe-sion molecule-1, was significantly elevated on CD4+ T

cells in VIR compared to the BDL group. This is consistentwith a previous report of the involvement of intercellularadhesion molecule-1 in syncytia formation and virusinfectivity and the increase in its expression on lym-phocytes in HIV infection [40].

Interestingly, the expression levels of two NK associatedreceptors on CD8+ T cells, CD16 and CD56 were signifi-cantly higher for LTNP than for BDL and VIR groups,though not all detected differences reached statistical sig-nificance (p = 0.0093 to 0.0871). CD56 is expressed on asubset of CD8+ T cells (mature cytolytic effector cells) andit has previously been suggested that the defective expres-sion of CD56 on these cells in HIV-infected individualscould contribute to the decreased peripheral blood T-cellcytotoxicity found in HIV infection [41]. These findings,together with ours, support the hypothesis that the CD8+T cells from LNTP may have stronger cytotoxic activitythan those from other HIV+ individuals. CD16 expressionon CD8+ T cells in HIV disease has not previously beenreported. However, increases in CD8 T cells expressing NKassociated receptors have been reported in melanomapatients, and these cells display an effector phenotype[42]. Similar changes may also occur in HIV patients, andthe implication of these changes needs further investiga-tion.

ConclusionDotScan antibody microarray technology enabled theidentification of 3 distinct HIV disease groups based on anextensive immunophenotypic characterization of thepatients' CD4+ and CD8+ peripheral blood T cells. Thisresearch not only confirmed previously reported findingsfrom flow cytometric investigations, but also demon-strated the power of the antibody microarray technology,by identifying 5 new cell surface antigens that may poten-tially be associated with HIV disease stages. Simultaneousscreening for a large number of cell surface antigensrevealed that changes in the expression of activationmarkers were more pronounced in CD8+ T cells, whereaschanges in the expression of cell membrane receptors forcytokines and chemokines were more pronounced inCD4+ T cells.

Since these changes were shown to be related to the dis-ease status, we suggest that the use of this technology willfacilitate further investigation of the causes and control ofHIV disease progression and eventually lead to a betterunderstanding of the pathogenesis of the disease. Ourstudy is the first to demonstrate how density of cell surfaceantigens can be efficiently exploited in an array manner inrelation to disease stages. This new platform of identifyingdisease markers can be further extended to study otherdiseases. Increasing patient group size should correspond-ingly improve the statistical significance of the observed



differences in antigen expression associated with diseasestage. A simplified protocol of direct purification of CD4+or CD8+ T cells from whole blood may also allow abroader diagnostic utility. Serial time course studies ofpatients during their disease progression should also pro-vide useful information on the modulation of cell surfaceantigens over time and could potentially identify newprognostic and therapeutic markers relevant to HIV dis-ease, enabling prediction of patient responsiveness totherapy.

MethodsPatient profilesBlood (20 ml EDTA) was obtained from 26 HIV+ individ-uals attending HIV clinic at the Westmead Hospital (Table1) and 5 HIV- healthy individuals from the Australian RedCross, Sydney. This study has been approved by the West-ern Sydney Area Health Services and all blood sampleswere obtained upon written informed consent. Thepatient groups were: (1) Healthy HIV- individuals (NEG;n = 5); (2) HIV+ individuals on HAART with "belowdetectable levels" of plasma viremia and classed aspatients controlling viremia with HAART (BDL; n = 11 forCD4; n = 10 for CD8; one CD8 sample was excluded as itfailed to meet the internal control criteria); (3) HIV+ indi-viduals on HAART with detectable plasma viremia (VIR; n= 9 for CD4; n = 10 for CD8; one CD4 sample wasexcluded as it failed to meet the internal control criteria);(4) Treatment naïve HIV+ long-term non-progressors(LTNP; n = 5), who have maintained high CD4+ T cellcounts (>500 cells/µl), with the average infection time of>20 years and natural control of plasma viremia to belowdetectable levels. One of the 5 LTNP patients (patient 26)in this category did show very low plasma viremia (128HIV RNA copies/ml of plasma), but was included becausethis patient met all the other selection criteria.

Purification of CD4+ and CD8+ T cellsA single blood sample (20 ml) was obtained from eachpatient. After separation of plasma, PBMC were isolatedby Ficoll-gradient centrifugation and then purified. CD4+and CD8+ T cells, respectively, were obtained by positiveisolation with antibody-conjugated magnetic beadsaccording to the manufacturer's instructions (Dynal Bio-tech, Oslo, Norway). Flow-cytometric analysis performedon separated CD4+ and CD8+ T cell populations demon-strated that in CD4+ T cell isolations 99.2% ± 0.165%(mean ± SD) of cells were single positive for CD4 marker,while 99.1% ± 0.128 (mean ± SD) of purified CD8+ cellswere single positive for CD8 marker [43]. Absence ofbinding of purified CD8+ T cells to the CD4 antibody andvice versa further confirmed that cross contamination wasnegligible and would not compromise assay specificity.

CD antibody microarraysMedsaic Pty. Ltd. (Eveleigh, NSW, Australia) provided theDotScanTM microarrays, prepared as previously described[44]. Monoclonal antibodies were purchased from the fol-lowing companies: Coulter and Immunotech from Beck-man Coulter (Gladesville, NSW, Australia), Pharmingen(BD Biosciences, North Ryde, NSW, Australia), BiosourceInternational (Applied Medical, Stafford City, QLD, Aus-tralia), Serotec (Australian Laboratory Services, Sydney,NSW, Australia), Sigma-Aldrich (Castle Hill, NSW, Aus-tralia), Biotrend, Biodesign and MBL (Jomar Diagnostics,Stepney, SA, Australia), Chemicon Australia (Boronia,VIC, Australia), Leinco Technologies (St. Louis, MO, USA)and Calbiochem (Merck, Kilsyth, VIC, Australia). Anti-body solutions were reconstituted as recommended, andstored in aliquots with 0.1% (w/v) BSA at -80°C;Pharmingen antibodies were generally stored at 4°C.Antibodies were used for making microarrays at concen-trations ranging from 50–1000 µg protein/ml.

Immunophenotyping of ex vivo purified CD4+ and CD8+ T cellsPurified CD4+ and CD8+ T cell populations were testedon antibody microarrays using DotScan technology aspreviously described [45]. Briefly, a 300 µL aliquot ofeither purified CD4+ or CD8+ cell suspension (= 4 × 106

cells) was incubated for 30 min on the microarray chip,after which unbound cells were removed by gentle immer-sion in PBS. Captured cells were fixed and imaged using aMedsaic DotReader™ and dot intensities were quantifiedfor each antigen in duplicate using Dotscan data analysissoftware on an 8-bit pixel grey scale from 0–255 thatreflects the level of expression of a particular antigen aswell as the proportion of cells expressing that antigen[45]. The limit of detection using the optical scanner isapproximately 100 cells/antibody dot. Each microarrayhas alignment dots, which establishes the location of eachdot on the array and also served as the internal control tomeasure the distribution of cells. The dot pattern obtainedis the immunophenotype of that population of leuko-cytes.

The main strength of antibody microarray is its capacity torapidly screen for a large number of antigens, producingan extensive immunophenotype using a relatively smallnumber of cells in a single assay. However, it would notprovide all of the information obtained by flow cytometrysuch as multiparameter analysis on single cells and levelof antigen expression per cell. When the same sample istested by the same operator on 3 different arrays (unpub-lished data), the coefficient of variation (CV) for bindingdensities tends to be low (8.3%) for dots of high density(>50 pixels), but higher (33.3%) for dots of low density(2–50 pixels). Reproducible dot binding patterns can be



achieved if a technically consistent standard assay proto-col is followed for all assays.

Statistical derivationsThe objective of this analysis was to identify antibody dotsshowing differential levels of binding of cells derivedfrom different disease categories, where the sample cate-gories had been established a priori. No fewer than 5 sam-ples per group were included for statistical analysis, asrequired for the application of Medsaic's standard bioin-formatics tools used in this study. Data were log trans-formed and log transformed antibody bindings have asymmetrical distribution which shows reasonable stabil-ity of variance with mean expression. Following transfor-mation, the distributional properties for individualantibodies were examined using box plots and kernel den-sity estimators. Differential expression was analysed on anantibody-by-antibody basis. Individual intra-group com-parisons were first carried out, followed by pairwise inter-group comparisons between the four study groups. The"Antibody Ranking" analysis of the array binding resultswas carried out as a one way analysis of variance, whichprovided p values and adjusted p values for each antibodyand ranked the antibodies in order of significance. P val-ues were adjusted using Holm's method, a conservativeapproach to maintain strong control of an inflated type Ierror rate [46]. Differential expression of antigens wasidentified by paired comparisons of the 4 study groups,with differences reaching statistical significance when theadjusted p value was less than 0.05.

AbbreviationsAbbreviations used in this paper:

BDL, below detection level;

HAART, highly active antiretroviral therapy;

LTNP, long-term non-progressor;

NEG, HIV seronegative individuals;

VIR, viremic patients.

Competing interestsThe author(s) declare that they have no competing inter-ests.

Authors' contributionsJQW fully performed the work, analyzed data and wrotethe paper. BW contributed to the writing. LB and JC ana-lyzed data, did statistical evaluation, contributed to thetechnology and the writing. JL and DED contributed tovital patient samples and immunological interpretation offindings. WBD, JZ, ALC, NKS designed the research

project, supervised this work and contributed to the writ-ing. All authors read and approved the final manuscript.

AcknowledgementsWe thank Dr. Mervyn Thomas for statistical analysis and assistance. We thank Dr. Choo Beng Chew for providing patient samples. Jing Qin Wu is thankful to the University of Sydney for the Australian Postgraduate Award for her PhD study and the Millennium Foundation for the top-up scholar-ship. This work was supported by grants from the AIDS Foundation Budget.

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